s2fieldsofstudy: Biology Medicine

text:
The Cysteine Protease Giardipain-1 from Giardia duodenalis Contributes to a Disruption of Intestinal Homeostasis

In giardiasis, diarrhoea, dehydration, malabsorption, weight loss and/or chronic inflammation are indicative of epithelial barrier dysfunction. However, the pathogenesis of giardiasis is still enigmatic in many aspects. Here, we show evidence that a cysteine protease of Giardia duodenalis called giardipain-1, contributes to the pathogenesis of giardiasis induced by trophozoites of the WB strain. In an experimental system, we demonstrate that purified giardipain-1 induces apoptosis and extrusion of epithelial cells at the tips of the villi in infected jirds (Meriones unguiculatus). Moreover, jird infection with trophozoites expressing giardipain-1 resulted in intestinal epithelial damage, cellular infiltration, crypt hyperplasia, goblet cell hypertrophy and oedema. Pathological alterations were more pronounced when jirds were infected intragastrically with Giardia trophozoites that stably overexpress giardipain-1. Furthermore, Giardia colonization in jirds results in a chronic inflammation that could relate to the dysbiosis triggered by the protist. Taken together, these results reveal that giardipain-1 plays a key role in the pathogenesis of giardiasis.

Introduction
The intestinal epithelia of humans and animals are crucial for maintaining gut homeostasis and a balanced relationship between gut microbiota, digesta and the mucosal immune system. The disruption of this barrier can lead to a range of disorders, such as inflammatory bowel disease, colonic cancer and/or infectious diarrhoeal diseases [1,2].
Intestinal pathogens, including viruses, bacteria and parasites, often alter or disrupt gut epithelial homeostasis [3]. For instance, the pathophysiology of giardiasis involves intestinal disturbances frequently associated with changes in the epithelium. Giardia duodenalis, a protistan parasite that often causes enteritis (giardiasis) in humans and other animals is cross-transmissible among species (depending on genotype or assemblage). Infection is acquired via the accidental ingestion of drinking water or food contaminated with cysts, and trophozoites excyst. Although motile, many trophozoites attach to the tips of epithelial cells in the small intestine (usually duodenum and jejunum) where they replicate by binary fission [4]. Although infections with this parasite can be asymptomatic, many are not, and giardiasis ensues as a consequence of an imbalance in the intimate host-parasite relationship [5][6][7][8][9][10].
Despite decades of research, the pathogenesis of giardiasis is still not fully understood. However, we do know that Giardia produces a range of virulence factors including cysteine rich surface proteins, tenascins and cysteine proteases [5,10,11]. Virulence factors produced by Giardia can trigger a broad range of mechanisms to disrupt the homeostasis of the intestinal mucosa. For instance, some cysteine proteases appear to cleave the intestinal epithelial cytoskeletal protein villin and degrade cytokines including IL8 and CXCL1, CXCL2, CXCL3, CCL2 and CCL20 [12][13][14]. Other secreted proteases can disrupt intracellular junctions in intestinal epithelial cells, and trigger dysbiosis in the gut [14][15][16][17]. Recently, we discovered a cysteine protease, giardipain-1-GL50803_14019; prior designation: CP2 [18]-that is released by trophozoites and has gelatinolytic activity and pro-apoptotic effects [10]. The characteristics of giardipain-1 suggested that the protease could play a key role in the pathogenesis of giardiasis. For this reason, we undertook the present exploration to assess the effect(s) of giardipain-1 on the intestinal epithelium in the jird (Meriones unguiculatus) model [19] and investigated chaWB_nges in intestinal homeostasis and microbiota in jirds infected with trophozoites expressing giardipain-1.

Giardipain-1 Induces Apoptosis in the Jird Duodenum
Apoptosis induction by G. duodenalis compromises the epithelial barrier [10]. Therefore, we evaluated the effect of giardipain-1 on the induction of apoptosis using the intestinal loop model. As shown in Figure 1, Intestinal epithelial cells (IEC) undergoing apoptosis (Green) were detected in both control and giardipain-1-treated loops. However, treatment with giardipain-1 increased the number of apoptotic cells at 6 h p.i. ( Figure 1H). Most apoptotic cells detected in giardipain-1-treated duodenal loops were epithelial cells that extruded from the surface of the villi, indicating that giardipain-1 triggers not only apoptosis but also anoikis in duodenal IEC ( Figure 1D,H, white arrows).

Giardipain-pAC-Transfected Trophozoites Enhance Epithelial Cell Damage in the Duodenum
Next, we evaluated the role of giardiapain-1 in the establishment of giardiasis. To do this, jirds were infected with 2.5 × 106 WB trophozoites that had a low expression of giardipain-1 (WB -); with wild-type trophozoites (WB + ) expressing giardipain-1; or with transfected trophozoites stably expressing giardipain-1 (WB giardipain-pAC trophozoites [10]. Uninfected jirds (n = 3 per date) receiving PBS were included as controls. Initially, we determined the expression of giardipain-1 in the three trophozoite lines used to infect the jirds, the WB giardipain-pAC, the WB + and the WB − . In these assays, a 25 kDa band recognized by mAb 1G3 was detected at high abundance in giardipain-pAC trophozoites extracts, with a medium abundance in WB + (wild type) and at a low level in WB − (Figure 2A(a)). Loading control used was G. duodenalis α-tubulin (Figure 2A(b)). In the same extracts from WB + and WB − and WB giardipain-pAC, trophozoites, the 25 kDa band exhibited medium, low and high gelatinolytic activity, respectively ( Figure 2B).
Plasmid detection was carried out in WB giardipain-pAC trophozoites cultured for for 15 to 21 days in the absence of puromicin and then for 10-20 days in the presence of this antibiotic to select the remaining trophozoites expressing the transfected plasmid. Cultures showed a positive growth demonstrating that indeed these trophozites still harboured the plasmid. Positive growth was also observed in axenized WB giardipain-pAC trophozoites recovered at days 7 and 21 p.i. from the intestine of jirds infected with WB giardipain-pAC trophozoites which were cultured in the presence of puromicin.

Giardipain-pAC-Transfected Trophozoites Enhance Epithelial Cell Damage in the Duodenum
Next, we evaluated the role of giardiapain-1 in the establishment of giardiasis. To do this, jirds were infected with 2.5 × 106 WB trophozoites that had a low expression of giardipain-1 (WB -); with wild-type trophozoites (WB + ) expressing giardipain-1; or with transfected trophozoites stably expressing giardipain-1 (WB giardipain-pAC trophozoites [10]. Uninfected jirds (n = 3 per date) receiving PBS were included as controls. Initially, we determined the expression of giardipain-1 in the three trophozoite lines used to infect the jirds, the WB giardipain-pAC, the WB + and the WB − . In these assays, a 25 kDa band recognized by mAb 1G3 was detected at high abundance in giardipain-pAC trophozoites extracts, with a medium abundance in WB + (wild type) and at a low level in WB − (Figure  2A(a)). Loading control used was G. duodenalis α-tubulin (Figure 2A(b)). In the same extracts from WB + and WB − and WB giardipain-pAC, trophozoites, the 25 kDa band exhibited medium, low and high gelatinolytic activity, respectively ( Figure 2B).  Next, we carried out histopathological examinations of the duodenal epithelia of jirds infected with either WB − , WB + or WB giardipain-pAC trophozoites at 14, 21, 90 or 180 days. Jirds that received PBS showed normal mucosal and epithelial architecture. However, at 14 days p.i., the presence of Giardia trophozoites increased inflammatory cell infiltrate and thickened the villi. The inflammatory infiltration in the mucosa was represented mainly by lymphocytes and plasma cells. Enterocytic hyperplasia at the edge of the villi and histological alterations at the tips of villi were also observed in the presence of the trophozoites. Interestingly, these changes were more prominent in the mucosa of jirds exposed to WB giardipain-pAC ( Figure 4M) as compared with those infected with WB + trophozoites ( Figure 4I). Less damage was evident in the duodenal epithelia from jirds infected with WB − trophozoites which exhibited low girdipain-1 expression ( Figure 4E). However, an assessment of trophozoite burdens in jirds showed no marked difference among the groups infected with WB giardipain-pAC, WB + and WB − trophozoites at day 14, the time at which patency peaks in jirds [19][20][21][22] (Figure 3). Plasmid detection was carried out in WB giardipain-pAC trophozoites cultured for for 15 to 21 days in the absence of puromicin and then for 10-20 days in the presence of this antibiotic to select the remaining trophozoites expressing the transfected plasmid. Cultures showed a positive growth demonstrating that indeed these trophozites still harboured the plasmid. Positive growth was also observed in axenized WB giardipain-pAC trophozoites recovered at days 7 and 21 p.i. from the intestine of jirds infected with WB giardipain-pAC trophozoites which were cultured in the presence of puromicin.
Next, we carried out histopathological examinations of the duodenal epithelia of jirds infected with either WB − , WB + or WB giardipain-pAC trophozoites at 14, 21, 90 or 180 days. Jirds that received PBS showed normal mucosal and epithelial architecture. However, at 14 days p.i., the presence of Giardia trophozoites increased inflammatory cell infiltrate and thickened the villi. The inflammatory infiltration in the mucosa was represented mainly by lymphocytes and plasma cells. Enterocytic hyperplasia at the edge of the villi and histological alterations at the tips of villi were also observed in the presence At 21 days p.i., the infiltration with lymphocytes and plasma cells persisted in the inflamed mucosa and that resulted in distortion of villi, characterized by a thickening due to inflammatory cell infiltration. Additionally, in some areas, the presence of a wider inter-villus space was observed, and a random shortening of the villi was also detected. Interestingly, distension and thickening of the tips of villi was associated with inflammatory cells and oedema, such that the villi appeared distorted. As for the observations made at 14 days, all of the histopathological changes were exacerbated when jirds were infected with WB pACgiardipain-1 trophozoites ( Figure 4N) compared with those infected with WB + trophozoites ( Figure 4J). As expected, WB − trophozoites induced less damage in the duodenal epithelia than those infected with either WB giardipain-pAC or WB + trophozoites ( Figure 4F). At 3 months p.i., the multifocal separation, the shortening and the distortion of the villi clearly persisted. However, at this time point, we also detected fusion of villi ( Figure 4G-I).
The thickening of the villi observed in the infected jird was likely due to oedema in the submucosa at the tip of the villus and inflammatory cell infiltration at the crypt base. Again, the changes were more prominent in jirds infected with WB giardipain-pAC trophozoites ( Figure 4O) compared with those infected with WB + trophozoites ( Figure 4K). Less damage was evident in the duodenal epithelia from jirds infected with WB − trophozoites ( Figure 4G). The infection of jirds with Giardia duodenalis trophozoites triggers microscopic alterations in the duodenum of these animals. Representative histological sections of the duodenum of control jirds or animals that were infected with WB − , or WB + or WB giardipain-pAC. Time p.i. are 14 days, 21 days, 3 months and 6 months. Panels A to D are sections of control animals displaying normal mucosa with no alterations. In Panels E to H are sections of the duodenum from jirds infected with WB − trophozoites, in panels I to L are section of duodenum from jirds infected with WB + trophozoites and in panels M to P are sections of jirds infected with WB giardipain-pAC trophozoites. Hypertrophy, shortening and fusion of villi (black arrow) as well as hyperplasia of the intestinal crypts (head arrow), oedema and inflammatory infiltrate (*) were observed in WB + infected jirds. The alterations were more severe at 6 months p.i. WB giardipain-pAC trophozoite induced similar histopathological alterations to the ones described for WB + trophozoites However, the changes were more severe and cryptitis (black ball) was observed at 6 months p.i. A lower number of globet cells were observed in WB − infected jirds. H&E stain, 300 µm and 100 µm scale bar. At 6 months p.i., the alterations in the mucosa, including the distortion and villus fusion, thickening linked to the lymphoplasmacytic infiltration, shortening of the villi and oedema persisted in all trophozoites-infected groups. The changes were more prominent in jirds infected with WB giardipain-pAC trophozoites ( Figure 4P) when compared with those infected with WB + trophozoites ( Figure 4L). Additionally, only in jirds infected with WB giardipain-pAC trophozoites ( Figure 4P), we detected the presence of distended crypts and cell detritus (proteinaceous material and inflammatory cells) in the lumen that was compatible with exudative/suppurative cryptitis. There was less damage in the intestinal epithelium in jirds infected with WB − trophozoites ( Figure 4H) than those with WB giardipain-pAC or WB + trophozoites, again indicating that giardipain-1 protein levels are important in the damage induced in the intestinal mucosa.
Overall, these findings indicate that Giardia trophozoites induce significant morphological alterations in the intestinal mucosa of jirds. Furthermore, the histological scores corroborated that there was more mucosal damage in jirds infected with WB giardipain-pAC trophozoites than in those infected with WB + or WB − trophozoites (Supplementary Figure S1A). Further, the number of globet cells per villi was higher in jirds infected with WB giardipain-pAC trophozoites than in those infected with WB + or WB − trophozoites (Supplementary Figure S1B).

Trophozoites Increase Epithelial Cell Proliferation in the Duodenum
Histological damages are often related to changes in cell homeostasis. Therefore, we evaluated cell proliferation in the mucosa of jirds infected with WB + or WB giardipain-pAC by detecting the presence of Ki67 and pHist3 [23]. Immunofluorescence staining of both proliferation markers was carried out in the duodenum of jirds infected with Giardia for 14 or 21 days or 3 months. As shown in Figure 5A

Giardia Induces Goblet Cell Hyperplasia
Increased proliferation of IEC during infection is often associated with hyperplasia of goblet cells [24] and it is known that giardiasis increases the number of goblet cells in the small intestine [25]. Thus, we analyzed the presence of goblet cells in the intestine of jirds infected with WB + or WB giardipain-pAC trophozoites. To this end, polysaccharides and mucus were analyzed by PAS-AB staining in the duodenum of jirds infected with trophozoites for 14 and 21 days and 3 and 6 months. As shown in Figure 6A-D in uninfected controls, the goblet cells were randomly distributed along the crypt-villus axis, and its numbers remained constant with age. In contrast, the number of goblet cells increased moderately in the duodenum of jirds infected with each trophozoite line, starting at 14 days p.i., but was significantly less in jirds infected with WB − trophozoites ( Figure 6E). The random distribution of goblet cells was maintained along the crypt axis in jirds infected with either WB − or WB + or WB giardipain-pAC trophozoites ( Figure 6E,I,M). Starting at day 21 p.i., the number of goblet cells in the intestinal mucosa of jirds infected with WB + and WB giardipain-pAC trophozoites increased significantly, ( Figure 6F,J,N) and their presence was more prominent at the crypt base of the villi in the proliferative compartment.

Giardia Induces Goblet Cell Hyperplasia
Increased proliferation of IEC during infection is often associated with hyperplasia of goblet cells [24] and it is known that giardiasis increases the number of goblet cells in the small intestine [25]. Thus, we analyzed the presence of goblet cells in the intestine of jirds infected with WB + or WB giardipain-pAC trophozoites. To this end, polysaccharides and mucus were analyzed by PAS-AB staining in the duodenum of jirds infected with trophozoites for 14 and 21 days and 3 and 6 months. As shown in Figure 6A-D in uninfected controls, the goblet cells were randomly distributed along the crypt-villus axis, and its numbers remained constant with age. In contrast, the number of goblet cells increased moderately in the duodenum of jirds infected with each trophozoite line, starting at 14 days p.i., but was significantly less in jirds infected with WB − trophozoites ( Figure 6E). The random distribution of goblet cells was maintained along the crypt axis in jirds infected with either WB − or WB + or WB giardipain-pAC trophozoites ( Figure 6E,I,M). Starting at day 21 p.i., the number of goblet cells in the intestinal mucosa of jirds infected with WB + and WB giardipain-pAC trophozoites increased significantly, (Figure 6F,J,N) and their presence was more prominent at the crypt base of the villi in the proliferative compartment.
At 3 months p.i., WB + trophozoites and WB giardipain-pAC trophozoites ( Figure  6K,O) induced more goblet cells with larger mucus vacuoles in the intestinal crypts of duodenum when compared with duodenum crypts of jirds infected with WB − trophozoites ( Figure 6G). Interestingly, in infected jirds in general, the number of goblet cells at the At 3 months p.i., WB + trophozoites and WB giardipain-pAC trophozoites ( Figure 6K,O) induced more goblet cells with larger mucus vacuoles in the intestinal crypts of duodenum when compared with duodenum crypts of jirds infected with WB − trophozoites ( Figure 6G). Interestingly, in infected jirds in general, the number of goblet cells at the apex of the villus was reduced. The numbers and distribution of the goblet cells in the intestinal mucosa of infected jirds was maintained for 6 months p.i. as compared with control jirds. However, when compare between the different trophozoite strains the number of goblet cells was higher when jirds were infected with WB trophozoites overexpressing pACgiardipain-1 ( Figure 6P) than those infected with WB + trophozoites ( Figure 6L) or with WB − trophozoites ( Figure 6H). These findings show clearly that giardipain-1 significantly increases the number of goblet cells in the duodenum of infected jirds. Subsequently, we explored the distribution of mucin 2 (MUC2), the main mucopolysaccharide produced by goblet cells in the intestine. In control jirds, MUC2 (green) was enriched in the cytosol of goblet cells randomly distributed along the crypt-villi axis (Figure 7A,D,G). The number of MUC2-positive cells along the crypt axis was constant, irrespective of jird age. Additionally, a faint layer of MUC2 on the apex and the epithelial brush border was readily detected (Figure 7A, arrow). However, the infection with WB + or WB giardipain-pAC trophozoites for 14 and 21 days and 3 months markedly thickened the layer of MUC2 on the luminal side and increased the number of MUC2-positive cells ( Figure 7B,C,E,F,H,I). The layer of MUC2 observed along the whole crypt-villi axis at 14 days p.i. suggested an active and relatively uniform secretion of the mucopolysaccharide by the goblet cells ( Figure 7B,C). However, after 21 days and 3 months p.i., the layer of MUC2 was concentrated at the crypt base, suggesting an active secretion by newlyformed goblet cells and an atrophy or exhaustion of superficially-located goblet cells ( Figure 7E,F,H,I). Importantly, at 3 months p.i., the luminal layer of MUC2 was more prominent in the duodenum of jirds infected with WB giardipain-pAC trophozoites than in jirds infected with WB + trophozoites. Taken together, these findings indicate that IEC proliferation gives rise to 'hypersecretory' goblet cells during giardiasis.

Giardia Triggers a Chronic Inflammatory Response in the Duodenum
The T-cell response triggered during giardiasis in the intestine can clear Giardia infection at around 6 weeks [26]. However, our results reveal a chronic inflammatory response in the duodenum of jirds infected with Giardia that extends beyond the 6-week window. Thus, we evaluated the presence of T cells in the duodenum mucosa of jirds infected with WB + or WB giardipain-pAC trophozoites for 14 and 21 days and 3 months. CD3 + cells were detected in the lamina propria of the intestinal crypts, and these cells were in close apposition with the basal membrane of IEC (Figure 8A,D,G). As expected, the number of CD3 + cells increased markedly in the duodenum mucosa of jirds infected with WB + or WB giardipain-pAC trophozoites for 14 and 21 days ( Figure 8B,C,E,F). However, the number of CD3 + cells remained elevated in the duodenal mucosa of jirds at 3 months p.i. when the Giardia infection had already been cleared ( Figure 8H,I). Importantly, in the duodenal mucosa of jirds infected with WB + or WB giardipain-pAC trophozoites, CD3 + cells were detected in the lamina propria along the crypt-villi axis ( Figure 8B,C,E,F,H,I), and these cells were enriched around the crypt base. Furthermore, a higher number of CD3 + cells were recruited into the lamina propria in jirds infected with WB giardipain-pAC trophozoites that those infected with WB + trophozoites, suggesting a role for giardipain-1 in this process. These results indicate that giardiasis triggers a strong chronic immune response mediated by CD3 + cells in the duodenum of jirds.

Giardia Triggers a Chronic Inflammatory Response in the Duodenum
The T-cell response triggered during giardiasis in the intestine can clear Giardia infection at around 6 weeks [26]. However, our results reveal a chronic inflammatory response in the duodenum of jirds infected with Giardia that extends beyond the 6-week window. Thus, we evaluated the presence of T cells in the duodenum mucosa of jirds infected with WB + or WB giardipain-pAC trophozoites for 14 and 21 days and 3 months. CD3 + cells were detected in the lamina propria of the intestinal crypts, and these cells were in close apposition with the basal membrane of IEC (Figure 8A,D,G). As expected, the number of CD3 + cells increased markedly in the duodenum mucosa of jirds infected with WB+ or WB giardipain-pAC trophozoites for 14 and 21 days ( Figure 8B,C,E,F). However, the number of CD3 + cells remained elevated in the duodenal mucosa of jirds at 3 months p.i. when the Giardia infection had already been cleared ( Figure 8H,I). Importantly, in the duodenal mucosa of jirds infected with WB + or WB giardipain-pAC trophozoites, CD3 + cells were detected in the lamina propria along the crypt-villi axis ( Figure 8B,C,E,F,H,I), and these cells were enriched around the crypt base. Furthermore, a higher number of CD3 + cells were recruited into the lamina propria in jirds infected with WB giardipain-pAC trophozoites that those infected with WB + trophozoites, suggesting a role for giardipain-1 in this process. These results indicate that giardiasis triggers a strong chronic immune response mediated by CD3 + cells in the duodenum of jirds.

Faecal Microbiota Transplanted to Jirds Infected with Giardia Triggers Epithelial Dysfunction and Changes in the Microbiome Composition
Based on previous results [15,16], we tested the hypothesis that the immune response elicited by giardiasis is mediated by changes in the microbiome. We approached this by transplanting faecal microbiota from jirds infected with WB giardipain-pAC trophozoites to a group of jirds kept as recipient controls and to another group of jirds infected with WB giardipain-pAC trophozoites. Following transplantation of faecal microbiota, microscopic examination of duodenal sections was carried out at different time points. At 21

Faecal Microbiota Transplanted to Jirds Infected with Giardia Triggers Epithelial Dysfunction and Changes in the Microbiome Composition
Based on previous results [15,16], we tested the hypothesis that the immune response elicited by giardiasis is mediated by changes in the microbiome. We approached this by transplanting faecal microbiota from jirds infected with WB giardipain-pAC trophozoites to a group of jirds kept as recipient controls and to another group of jirds infected with WB giardipain-pAC trophozoites. Following transplantation of faecal microbiota, microscopic examination of duodenal sections was carried out at different time points. At 21 p.i., control jirds had a normal mucosal and intestinal epithelial structure ( Figure 9A). However, the intestinal villi in jirds inoculated with microbiota from WB giardipain-pAC treated animals displayed villi thickening due to inflammatory infiltration (mainly lymphocytes) in the submucosa ( Figure 9B). Additionally, when the microbiota was transplanted in the group of jirds infected with WB giardipain-pAC, the changes in the intestinal epithelium were exacerbated. In those jirds, the villi were shortened, distorted and fused; they were also thickened, likely due to infiltration of predominantly lymphocytes and plasma cells. This thickening extended to the base of the crypts and was consistent with crypt hyperplasia ( Figure 9C). The histochemical analysis of duodenal sections obtained at 21 days p.i. from jirds inoculated with microbiota and then infected with WB giardipain-pAC trophozoites showed that goblet cells were more numerous, mainly in the crypts, contrasting lower numbers at the apices of villi ( Figure 9C). In microbiota giardipain-pAC treated group, there was a slight increase in the number of positive cells in the crypts ( Figure 9B) compared with the PBS-treated control group ( Figure 9A). p.i., control jirds had a normal mucosal and intestinal epithelial structure ( Figure 9A). However, the intestinal villi in jirds inoculated with microbiota from WB giardipain-pAC treated animals displayed villi thickening due to inflammatory infiltration (mainly lymphocytes) in the submucosa ( Figure 9B). Additionally, when the microbiota was transplanted in the group of jirds infected with WB giardipain-pAC, the changes in the intestinal epithelium were exacerbated. In those jirds, the villi were shortened, distorted and fused; they were also thickened, likely due to infiltration of predominantly lymphocytes and plasma cells. This thickening extended to the base of the crypts and was consistent with crypt hyperplasia ( Figure 9C). The histochemical analysis of duodenal sections obtained at 21 days p.i. from jirds inoculated with microbiota and then infected with WB giardipain-pAC trophozoites showed that goblet cells were more numerous, mainly in the crypts, contrasting lower numbers at the apices of villi ( Figure 9C). In microbiota giardipain-pAC treated group, there was a slight increase in the number of positive cells in the crypts ( Figure 9B) compared with the PBS-treated control group ( Figure 9A). Figure 9. Microscopic alterations in sections of the duodenum of the animals treated with fecotransplants, then infected with WB giardipain-pAC trophozoites and euthanized at 21 days p.i. Representative histological and histochemistry sections of the duodenum of jirds that received the microbiota of previously infected jirds and then were infected with WB giardipain-pAC. Sections are from: (A) control animals displaying normal mucosa with no alterations. (B) jirds given microbiota from animals infected with WB giardipain-pAC trophozoites; in these animals slight thickening of villi was observed, (C) animals that received microbiota and that were infected with WB giardipain-pAC. In these atrophy, fusion of villi (red arrow) as well as hyperplasia of the intestinal crypts (head arrow) and inflammatory infiltrate (*) were observed by H&E stain, 300 μm and 100 scale bar. PAS-AB+ stained of sections from: (D) control animals in which the normal population of goblet cells in intestinal villi was observed. (E) jirds that were given microbiota from animals previously infected with WB giardipain-pAC trophozoites; in these sections a slight increase in goblet cells was observed and (F) animals given microbiota and then infected with WB giardipain-pAC. In these a marked increase in goblet cells was observed = arrow. Scale bar = 300 μm and 100 μM.
Alterations in the intestinal microbiota that associate with intestinal inflammation [27] were studied. To determine whether Giardia infection impacts the microbiome in jirds, DNA from faeces were analyzed using the Illumina Miseq platform. The results showed Figure 9. Microscopic alterations in sections of the duodenum of the animals treated with fecotransplants, then infected with WB giardipain-pAC trophozoites and euthanized at 21 days p.i. Representative histological and histochemistry sections of the duodenum of jirds that received the microbiota of previously infected jirds and then were infected with WB giardipain-pAC. Sections are from: (A) control animals displaying normal mucosa with no alterations. (B) jirds given microbiota from animals infected with WB giardipain-pAC trophozoites; in these animals slight thickening of villi was observed, (C) animals that received microbiota and that were infected with WB giardipain-pAC. In these atrophy, fusion of villi (red arrow) as well as hyperplasia of the intestinal crypts (head arrow) and inflammatory infiltrate (*) were observed by H&E stain, 300 µm and 100 scale bar. PAS-AB+ stained of sections from: (D) control animals in which the normal population of goblet cells in intestinal villi was observed. (E) jirds that were given microbiota from animals previously infected with WB giardipain-pAC trophozoites; in these sections a slight increase in goblet cells was observed and (F) animals given microbiota and then infected with WB giardipain-pAC. In these a marked increase in goblet cells was observed = arrow. Scale bar = 300 µm and 100 µM.
Alterations in the intestinal microbiota that associate with intestinal inflammation [27] were studied. To determine whether Giardia infection impacts the microbiome in jirds, DNA from faeces were analyzed using the Illumina Miseq platform. The results showed that, when faecal transplants were given to jirds, there was a reduction in relative abundance of members of the families Alcaligenaceae and S24-7, while in jirds that received the microbiota and were infected with WB giardipain-pAC trophozoites, there was a reduction of F16 and an increase in the relative abundance of members of the Enterococcaceae, and Aerococcaceae was detected ( Figure 10A). Based on Bray-Curtis dissimilarities, no significant effect was observed on the bacteria community in faecal samples from jirds given microbiota or in jirds that received microbiota and had been infected with WB giardipain-pAC trophozoites ( Figure 10B). However, the heatmap did show significant changes in the bacterial taxa ( Figure 10C) between samples from jirds which received microbiota or those receiving microbiota and were infected with WB giardipain-pAC trophozoites. In the latter jirds, we observed a decrease in species of Sutterella (−3.45) and Oscillospira (−4.60). Interestingly, when this group of jirds was compared with the control group, a decrease in Ruminoccoccus gnavus (−6.23) and Oscillospira (−4.86) was observed. Thus, infection by Giardia trophozoites that overexpress giardipain-1 induce temporal disruption in the mucosa besides to impact in impaired microbiome eubiosis resulting in the inflammatory response observed in the infected animals.  Figure 10. Determination of changes in the microbiota (Mb) composition of jirds that received different treatments and were euthanized at 21 days p.i. Composition of the microbiota in jird feces that received different treatments was determined by 16S ribosomal RNA gene amplicon sequencing. The data were compared between the control group (PBS) and the group of animals that was given microbiota from giardipain-pAC infected animals and then were infected with WB giardipain-pAC trophozoites. (A) Average relative microbial abundance at the family levels in the different groups. (B) Variation in beta-diversity of jird gut bacterial communities based on Bray-Curtis dissimilarities. (C) Heat map for relative abundance at species level with significant changes.

Discussion
Here, we showed that giardipain-1 triggers apoptosis and an extrusion in epithelial cells (anoikis). This type of cell death occurs when epithelial cells lose intercellular contacts and undergo extrusion from the monolayer [28]. The present findings strongly support previous results indicating that giardipain-1 can induce apoptosis in epithelial cells Figure 10. Determination of changes in the microbiota (Mb) composition of jirds that received different treatments and were euthanized at 21 days p.i. Composition of the microbiota in jird feces that received different treatments was determined by 16S ribosomal RNA gene amplicon sequencing. The data were compared between the control group (PBS) and the group of animals that was given microbiota from giardipain-pAC infected animals and then were infected with WB giardipain-pAC trophozoites. (A) Average relative microbial abundance at the family levels in the different groups. (B) Variation in beta-diversity of jird gut bacterial communities based on Bray-Curtis dissimilarities. (C) Heat map for relative abundance at species level with significant changes.

Discussion
Here, we showed that giardipain-1 triggers apoptosis and an extrusion in epithelial cells (anoikis). This type of cell death occurs when epithelial cells lose intercellular contacts and undergo extrusion from the monolayer [28]. The present findings strongly support previous results indicating that giardipain-1 can induce apoptosis in epithelial cells [29,30] by targeting proteins at the tight and adherens junctions [10]. Low rates of apoptosis/anoikis in the intestinal epithelium eliminate aged or ravaged cells and maintain intestinal epithelial homeostasis [31]. Thus, our findings indicate that giardipain-1 affects epithelial homeostasis. The effect of giardiapain-1 is rapid and likely transient, given that in the intestinal mucosa the restoration/compensation mechanisms in the epithelium are activated immediately following infection, and is plausible that for that reason Giardia is rapidly eliminated from the gut. However, by destroying intercellular junctions and altering epithelial barrier functions, giardipain-1 might trigger the chronic effects that are associated with giardiasis, including the persistent pathological changes seen in the intestinal epithelium and/or the adaptive immune responses [15,32]. In fact, in our experiments the damage to the superficial mucosa of the small intestines of jirds infected with WB giardipain-pAC trophozoites was similar to that seen in people with celiac disease [33] or inflammatory bowel disease (IBS) [34]. Such damage includes the loss of normal villus structure, with a reduction in villus height, a marked enlargement and lengthening of crypts, and inflammatory cell infiltration with an increase density of intraepithelial lymphocytes [35]. Therefore, we propose that treatments aimed at inhibiting giardiapain-1 could help prevent various disorders associated with Giardia duodenalis infection, including IBS.
The acute pathological changes associated with the trophozoites included increased lymphoplasmacytic infiltration in the villi, fused villi, and changes in the cytoarchitecture of the villi; many of these alterations extended into the chronic phase of the infection. In this context, the infection of jirds with trophozoites overexpressing giardipain-1 potentiated acute and chronic inflammatory effects induced by normal trophozoites, indicating that giardiapain-1 contributes markedly to the pathogenesis of giardiasis. Our results likely explain villus atrophy, intestinal crypt hyperplasia and increased numbers of immune cells in the mucosa of patients with giardiasis, observed in previous works [36][37][38][39].
An important observation in jirds infected with the different trophozoite lines was the increase in number and size of goblet cells and the presence of luminal mucus in the intestinal crypts. This observation accords with previous observations of goblet cell hyperplasia associated with infections with Trichinella spiralis [36,37], Eimeria [38], Nippostrongylus brasilensis [39,40], Trichuris muris [41,42] and Giardia [43][44][45]. Here, we speculate that mucus hypersecretion is a compensatory mechanism, aimed at repairing barrier function of the intestinal epithelium. However, this could also act as a defense mechanism given cysteine protease activity has been linked to mucin degradation and depletion, and the alteration of mucin gene expression in Giardia [46,47]. Given that giardipain-1 stimulates epithelial removal and proliferation, we cannot exclude the possibility that proteolytic processing of the intercellular junctions by giardipain-1 enhances epithelial cell turnover and leads to a disruption of the epithelial barrier. Overall, our results indicate that proteolytic activity mediated by giardipain-1 triggers epithelial plasticity in the gut. Further work is needed to investigate role of signaling pathways in the induction of goblet cell hyperplasia during giardiasis.
Previous studies have shown that Giardia proteases can alter the composition of the intestinal microbiota and promote the formation of pathobionts [15,48,49]. Given that the intestinal microbiome protects the host against invasion by pathogens and against the overgrowth of pathobionts [50,51], this process is likely important in the pathogenesis of giardiasis. Indeed, the colonisation of pathobionts has been linked to the induction of inflammatory processes associated with the destruction of the epithelial barrier [52]. Further, it has been reported that the disruption of the microbiota during giardiasis can predispose to gastrointestinal disorders [15]. Interestingly, in our work, the alteration of bacterial taxa, such as a reduction in Sutterella, Rimunococcus gnavus and Oscillospira, in jirds infected with trophozoites, was similar to that observed in various inflammatory disorders of the small intestine [53][54][55]. On the other hand, the increase in members of the Enterococcaceae and Aerococcaceae may be due to alterations of the mucosal layer, together with changes in the innate and acquired immune responses, leading to an increase of these commensal microbiota as a consequence of dysbiosis in this model system. Their transcytosis through M cells, inducing IgA and IL 22 cytokines, as observed in mammalian hosts [56], could be a critical response to stimulate dendritic cells. Further, it has been reported that infection with the Giardia duodenalis strain GS causes systemic dysbiosis of commensal aerobic and anaerobic bacteria in the small intestine, specifically an increase in Proteobacteria and a decreased in Firmicutes and Melainabacteria [32], which can also be involved in post-infection complications associated with Giardia infection. Taken together, this information suggests that the microbiota play a key role during and after infection with Giardia, and that alterations in the microbiome could be linked to the development of some chronic inflammatory or immunological disorders [54].
In conclusion, giardipain-1-induced alterations seen in the intestinal epithelial cells and in the microbiome contribute to a better understanding of the pathogenesis of giardiasis and its associated complications.

Plasmid Detection in WB Giardipain-pAC Trophozoites by Puromycin-Selection
WB giardipain pAC trophozoites were cultured in the absence of puromycin for 15 or 21 days, after which puromycin at 100 µM was added and parasite growth was determined over a period of one week. In vivo, WB giardipain pAC trophozoites were collected from the small intestines of infected jirds at days 7 and 21. Then, trophozoites obtained were axenised, cultured in the presence of puromycin at 100 µM and the trophozoite growth determined.

Assessing the Effect of Purified Giardipain-1 on Intestinal Loops
Male jirds (8-10 weeks of age), maintained in a purposely-designed laboratory using a standard day/night light cycle and provided with a commercial diet and water ad libitum. All experimentation was approved by the Internal Committee for Care and Use of Laboratory Animals guidelines (CICUAL). The intestinal loop model was as described previously [59]. Briefly intestinal loops of jirds we injected with 32 µg of purified giardipain-1 in 200 µL of Dulbecco s Modified Eagle Medium (DMEM). Control animals received the same volume of DMEM only. Then apoptosis in the intestinal epithelium was determined at 4 and 6 h after giardipain-1 injection using the TUNEL assay [60].

Analysis of Giardipain-1 Expression in WB Giardipain-pAC, WB + and WB − Trophozoites by Western Blot, and Detection of Giardipain-1 Protease Activity by Zymogram Analysis
WB giardipain-pAC, or WB + or WB − trophozoites (10 × 10 6 ) were pelleted, suspended in 500 µL of PBS and sonicated in an ice bath at 60% amplitude. Three pulses were initially applied and then 20 µL of 1% Triton X-100 were added followed by two more pulses. Finally, the suspension was centrifuged at 4226× g for 10 min. Protein concentration was determined using the Pierce®Protein Assay Kit (Thermo Scientific) as recommended by the manufacturer. Then, samples (30 µg of protein each) were separated by SDS-PAGE (12%) under denaturing conditions. In this assay α-tubulin was used as loading-control. Gels were transferred to PVDF membranes for 1.5 h at 300 mAmp at 10 • C, after which the membranes were blocked with 5% (w/v) non-fat dried milk for 1 h at room temperature (24 • C) under constant agitation. Membranes were washed three times with TBS-T 0.1% and incubated with either IG3 monoclonal antibody (1:500) in 5% non-fat dried milk/TBS-T 0.1% overnight at 4 • C or with monoclonal anti-α-tubulin antibody (TAT1 control; 1:15,000; kindly donated by Dr Keith Gull from Oxford University, UK) for 12 h. Thereafter, the probed membranes were washed three times for 1 h at 24 • C and then incubated with 1: 10,000 goat anti-mouse IgG coupled to HRP in TBS-T as secondary antibody (GE-Healthcare). Membranes were reacted with ECL Plus Lightning ® Western kit (Perkin Elmer Inc., Waltham, MA, USA) and photographed using a UVP High Performance Ultraviolet Transilluminator fitted with Launch DocItLS software.
For zymograms, SDS-PAGE was performed using 12% polyacrylamide gels co-polymerized with 0.2% gelatin (Sigma); 30 µL of each total protein extracts under reducing conditions were loaded per lane and proteins were separated at 100 V for 2 h. The gels were further incubated in water containing 2.5% (v/v) Triton X-100 for 1 h and then washed 3 times in distilled water. To identify bands with proteolytic activity, the gels were incubated for 16 h at 37 • C in acetate buffer (pH 5.5) containing 1 mM DTT (constant agitation) and then washed 3 times with distilled water. Gels were stained with Coomassie blue (BioRad) for 2 h at 37 • C, washed with distilled water and incubated in 5% acetic acid /40% methanol (v/v). Gels were photographed as for Western blots.

Infection of Jirds and Faecal Microbiota Transplantation (FMT)
Animals were experimentally infected by gavage with 2.5 × 10 6 of WB − or the same number of WB + or WB giardipain-pAC trophozoites in 500 µL of phosphate-buffered saline (PBS). For transplantation, fresh faeces were collected from jirds infected with WB giardipain-pAC-transfected trophozoites. Pooled faeces were homogenized in PBS and diluted to 0.5 g/mL, and 150 µL of suspension were given by gavage to the of recipient jirds. Three independent experiments were done using the different Giardia cell lines employing three animals per group (including uninfected controls).

Monitoring the Number of Trophozoites in the Small Intestine of Jirds Infected with WB − or with WB + or with WB Giardipain-pAC Trophozoites
Jirds infected with either WB − , WB + or WB giardipain-pAC were euthanized at 14 days post infection, and the entire small intestines obtained from each animal were removed and placed in PBS. Then, the intestinal tissues were incubated for 45 min in cold PBS with constant agitation to dislodge the trophozoites from the intestinal mucosa. Trophozoites in the suspension were pelleted by centrifugation at 700× g for 5 min, and the pellet was washed twice with PBS. Finally, the pellet was resuspended in 20 µl of cold PBS, and the trophozoites were counted in a haemocytometer.

Histopathological Examination
Jirds were euthanized and the duodenum was removed and "Swiss rolls" prepared [61]. For immunofluorescence, the intestinal tissue was embedded in optimal cutting temperature (OCT) medium (Tissue-Tek) and snap frozen at −70 • C until use. For immunohistochemistry, the tissue was immersed in 4% formalin (buffered with PBS; pH 7.2) and paraffin-embedded sections (3 µm) were prepared. Conventional histology with haematoxylin-eosin (H&E) or Alcian blue/periodic acid-Schiff (AB/PAS) stained sections were scanned using a slide scanner (Scan Scope CS2, Leica, Nussloch, Germany) and analyzed using Aperio Im-ageScope software (Aperio). The lengths and widths of villi were measured and the number of goblet cells per villus counted in 50 fields using a 100× magnification. Histopathological changes were scored as follows: 1: minimal and focal inflammatory cell infiltrate in the mucosa, with an intact epithelial layer; 2: mild, diffuse inflammatory cell infiltrates in mucosa and submucosa. 3: moderate, diffuse inflammatory cell infiltrates in mucosa and submucosa, with distorted villous structure; 4: moderate, crypt hyperplasia, fusion villi and diffuse inflammatory cell infiltrates in mucosa and submucosa. were incubated for 1 h at room temperature, and coverslips mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). TUNEL staining was performed using the In Situ Cell Death Detection Fluorescein kit (Roche Diagnostics, Rotkreuz, Switzerland), according to the manufacturer's protocol. Images were collected using a confocal laser scanning microscope (TCS SP8, Leica, Wetzlar, Germany).

Immunohistochemistry
Deparaffinated and hydrated tissue was blocked for 5 min with 5% hydrogen peroxide, heated for antigen retrieval with Diva Decloaker (Biocare, Tijuana, Mexico) and blocked with Rodent Block R (Biocare, Mexico). Then, the sections were incubated with anti-CD3 (1:1000) (Biocare, Mexico) for 30 min at RT, and revealed with Betazoid DAB Chromogen and Betazoid DAB substrate buffer (Biocare, Mexico). Tissue sections were then incubated in haematoxylin, dehydrated and cover-slipped for microscopic examination.

Bacterial DNA Isolation and Illumina-Based Sequencing of 16S Amplicons
Stool samples were collected from each control and infected jirds. Genomic DNA was extracted from 100 mg faeces using a Pure LinkTM Microbiome DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to instructions. DNA was stored at −20 • C until processing.
Amplicon sequencing libraries were prepared by two-step PCR; 16S nuclear ribosomal RNA gene amplicons were prepared for the Illumina Miseq ® sequencing (Part # 15044223 Rev. B) according to the standard protocol provided by Illumina ® . First, bacterial 16S rDNA was amplified using primers S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 (Klindworth et al., 2013) in triplicate, pooled, purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany), and quantified using a Qubit 2.0 dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA). PCR products were 'normalized' to 5 ng/µL and then barcoded with oligonucleotides matching those from the Nextera XT Index Kit (Illumina, SD, USA). Successful amplification of the target 464 bp amplicon, which spans the V3 and V4 regions of the 16S rRNA gene, was verified by electrophoresis in 0.8% agarose gels [62]. Paired-end 16S amplicons were quantified, normalized and pooled. Second, amplicons were sequenced using V2 MiSeq reagent kit (500-cycles); multiplexed libraries were adjusted to a final concentration of 12 picomolar (pM), and contained 30% PhiX Control V3 library (Illumina, SD, USA).

Inferring Microbial Operational Taxonomic Units (OTUs)
The 1,192,734 reads across the V3-V4 regions produced by Illumina sequencing were trimmed, quality filtered, and the potential chimeric reads removed and demultiplexed by eliminating low abundant operational taxonomic units (OTUs), 571 308 sequences were retained for subsequent analysis. Taxonomic assignment was done using the QIIME2 suite [63]. Taxonomic grouping was conducted using data in the GreenGenes database, with the sequences grouping at 99% similarity. Alpha-and beta-diversities were computed using the vegan R package [64].
The ordering of samples from different groups was based, with (dis)similarities visualized by non-metric distance scaling (NMDS) and a permutational multivariate analysis of variance (PERMANOVA) using distance matrices (vegan:adonis) was performed. Alphadiversity parameters and the occurrence of bacterial families were tested for all groups using a non-parametric Kruskal-Wallis test, adjusted for multiple comparisons by the Benjamini-Hochberg method using DESeq tool. For this analysis, in the case of a significant difference (p < 0.05), pairwise group comparisons (infected versus control) were performed using the Mann-Whitney test corrected for multiple testing, resulting in the indicated p values. From the results from DESeq2, heat maps were prepared to display changes in mean abundance (log2 fold changes or logarithm base two of the change) for the different clades, and comparisons between/among groups. Each row of the heat maps corresponded to a clade and each column to a comparison between conditions.

Statistical Analysis
Data were expressed as a mean ± standard error of the mean (SEM); comparisons were made between groups using a one-way ANOVA, with a Tukey's post-hoc test, where p < 0.05 was considered significant. Statistical comparisons were made in GraphPad Prism 5.0 for Windows (San Diego, CA, USA).


Longitudinal imaging of individual olfactory sensory neurons in situ

Olfactory sensory neurons are found deep within the nasal cavity at a spatially restricted sheet of sensory epithelium. Due to their location behind the nasal turbinates, accessing these cells for physiological measurements in living animals is challenging, and until recently, not possible. As a further complication, damage to the overlying bone on the dorsal surface of the snout disrupts the negative pressure distribution throughout the nasal cavities, which fundamentally alters how odorants are delivered to the sensory epithelium and the inherent mechanosensory properties of olfactory sensory neurons in live animals. The approach described here circumvents these limitations and allows for optical access to olfactory sensory neurons in mice across time scales ranging from days to months.

Introduction
Olfactory transduction begins when volatilized odorant molecules are inhaled through the nasal cavities, where they bind to olfactory receptor proteins that are found on the cilia of olfactory sensory neurons (OSNs). The mouse olfactory epithelium contains ∼10 7 individual OSNs that can be broadly classified into one of ∼1,100 subtypes based on their receptor protein expression (Zhang and Firestein, 2002;Kawagishi et al., 2014). Each OSN expresses a single receptor protein subtype that is selective to a chemical functional group on an odorant molecule (Figures 1A,B). Odorant molecules each activate a combinatorial pattern of OSNs depending on their ligand sensitivity, which then signals the presence of functional groups in the environment.
All OSN that express the same receptor protein project an axon toward one of two common structures on the surface of the olfactory bulb called glomeruli (Mombaerts, 2006;Francia and Lodovichi, 2021). The distribution of olfactory sensory neuron subtypes in the dorsal aspect of the olfactory epithelium is consistent with the patterning of glomeruli on the dorsal surface of the olfactory bulb (Figures 1C,D; Ressler et al., 1993;Murthy, 2011). Many studies have measured the collective activity of olfactory sensory neurons to OSNs at their axon terminals because the approach provides an excellent signal-to-noise ratio and population readout (Meister and Bonhoeffer, 2001;Wachowiak and Cohen, 2001;Fletcher et al., 2009;Soucy et al., 2009;Albeanu et al., 2018;Zak et al., 2018); however, the ability to measure stimulus encoding properties at the single-cell level is not possible due to the tight packing of sensory neuron axons within the glomerular neuropil. Other studies of OSN function and physiology have avoided these limitations by excising tissue for ex vivo preparations (Ma et al., 1999;Ma and Shepherd, 2000;Xu et al., 2020), or by recording from a limited number of cells by puncturing the overlying bone (Duchamp-Viret et al., 1999). Both approaches are incompatible with measuring neural responses in the context of natural respiration or at the singlecell level.
This protocol describes an adapted experimental preparation that allows for repeated in vivo measurements of sensory neuron activity in freely breathing animals. The approach combines a thinned-skull technique (Yang et al., 2010) with chronic coverslip implantation that allows for clear visualization of neuronal responses on a scale of weeks to months. The preparation is best suited for multiphoton imaging due to the deep embedding of sensory neurons in the olfactory epithelium.

Experimental model and subject details
Adult (>8 weeks) OMP-GCaMP3 mice of both sexes were used in this study. Three separate animals were used for epithelial windows and the collection of example data. Mice were acquired from in-house breeding stocks and maintained within Harvard University's Biological Research Infrastructure for the duration of the study. All animals were between 20 and 30 g before surgery and singly housed following any surgical procedure. All mice used in this study were housed in an inverted 12-h light cycle at 22 ± 1 • C at 30-70% humidity and fed ad libitum. All the experiments were performed under the guidelines set by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at Harvard University. Thinned-bone cranial windows were prepared as described below and animals were allowed to recover for at least 3 days before the initiation of imaging experiments.

Multiphoton imaging
A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20X, 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4-8 frames/s. Fields of view ranged from 180 × 180 µm to 720 × 720 µm. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 µm. Image acquisition and scanning were controlled by custom-written software in LabVIEW (National Instruments). Emitted light was routed through two dichroic mirrors (680dcxr, Chroma, and FF555-Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu).

Odor stimulation
The monomolecular odorant Methyl tiglate (Penta Manufacturing) was used as a stimulus and delivered by a custom-built 16-channel olfactometer that was controlled by custom-written software in LabVIEW. The initial concentration of each odor was 16% (v/v) in mineral oil then further diluted 16 times with air. For all experiments, the airflow to the animal was held constant at 100 mL/min and odors were injected into a carrier stream. Respiration signals were collected using an airflow sensor (Honeywell AWM3300V; Bolding and Franks, 2017;Grimaud and Murthy, 2018).

Data analysis
Images were processed using both custom and available MATLAB (MathWorks) scripts. Motion artifact compensation and denoising were done using NoRMCorre (Pnevmatikakis and Giovannucci, 2017). The CaIMaN CNMF pipeline (Pnevmatikakis et al., 2016) was used to select and demix ROIs. ROIs were further filtered by size and shape to remove merged cells. dF/F values were calculated using the mean of a baseline period of at least 20 frames. To identify OSNs that were significantly odorant-modulated, three points centered on the peak dF/F signal after odorant delivery were averaged. OSNs were classified as significantly odorant-modulated if their peak averaged response exceeded 2.5 standard deviations of the baseline noise.

Surgical procedures
Cranial windows are prepared over a 2-day period. The first day consists of collecting necessary equipment and preparing the animal for surgery. The main surgery can typically be completed in 60-90 min the following day.
Day prior to surgery 1. Make a silicone whip by dipping an old drill bit in silicone sealant and withdrawing to leave a taper, as described by Shih et al. (2012). 2. Make coverslips. Using a diamond-tipped scribe, score a large coverslip in a checkerboard yielding rectangles 3 mm × 2 mm. From the checkerboard, break off individual sections to be used. Store in ethanol overnight. The remaining coverslip sections can be saved for future use. 3. Give the experimental animal a single dose of carprofen (5 mg/kg) subcutaneously to help prevent inflammation during surgery and recovery.
Day of surgery 1. One hour prior to surgery, give the animal a second dose of carprofen, (5 mg/kg) subcutaneously. 2. Collect consumables ( Table 1) and sterilize instruments ( Table 2) where possible using a steam autoclave or hot bead sterilizer. 3. Cut small corners of gelfoam from a larger sheet and place them in 1 mL of artificial cerebrospinal fluid (aCSF). 4. Sterilize the surgical area and stereotax with ethanol and cover the area with a drape. 5. Anesthetize the animal using a mixture of ketamine and xylazine (100 and 5 mg/kg, respectively). Before proceeding, verify anesthesia depth by toe pinch.
6. Place petroleum jelly on the eyes to prevent them from drying out. 7. Remove the hair from the crown of the head to the tip of the snout using electric clippers. Use an ethanol pad to clear any remaining hair or debris. 8. Place the animal in the stereotaxic device and ensure that the head is held firmly in place by ear bars by gently pressing on the snout. 9. Use the scalpel blade to make an incision at the midline from the crown of the head to the nares. Do not cut the tissue surrounding the nares. 10. Remove the flaps of skin on either side of the midline cut from the crown of the head toward the ocular sinus, then to a position just posterior of the nares. Take special care not to damage arteries that surround the eyes when removing this skin. If bleeding occurs, use small pieces of gelfoam soaked in aCSF to induce clotting and cellulose spears to absorb any remaining fluids. 11. Use a cotton-tipped applicator to remove any fascia or connective tissue covering the skull and snout of the animal. A scalpel blade may be used on older animals where more tissue is present.  12. Place a small drop of aCSF on the bone covering the snout in the area to be thinned (see Figures 2A,B). The thinned area should be directly anterior to the frontonasal suture, between the internasal suture and nasal-maxillary suture where the bone plate begins to widen. Allow the aCSF to soak in and soften the bone for 1-2 min. 13. Under the stereoscope, with a fresh drill bit, begin to thin the bone over the olfactory epithelium by lightly pressing the bit to the skull and moving in slow circular motions. 14. Periodically, use cellulose spears and more aCSF to clean the thinned area. 15. Check the thinning progress by placing a small amount of aCSF on the thinned area. If the bone remains opaque when moistened, continue with the drill bit. If the bone is transparent, place a fresh blade on the scalpel. 16. In sweeping motions from posterior to anterior, use the scalpel blade to gently shave the remaining bone. 17. Continue shaving the bone until it becomes soft and flexible like cellophane. Often, when the bone has reached the desired degree of thinness, the bone will begin to form a rounded upward projecting bubble from pressure within the nasal cavity. Special care needs to be taken not to puncture the thinned bone. Punctured bone will ruin the preparation by affecting airflow through the nasal cavity and allowing the glue to be absorbed below the bone in subsequent steps. 18. Clean the thinned portion of the skull with aCSF and remove any debris. 19. Create a slurry by mixing a small amount of tin oxide and aCSF on the thinned section of bone. 20. Use the silicone whip drill bit to polish the thinned portion of the skull with the tin oxide slurry, again taking care not to damage the thinned section. The polishing technique smooths the bone surface and provides a tight junction for the coverslip (step 22), while also reducing the bone opacity. 21. Again, clean the skull and remove any debris. Allow the skull to dry completely before moving to the next step. 22. Place a drop of cyanoacrylate on the thinned portion of the skull and place one of the coverslips on the drop of glue. The cyanoacrylate should spread evenly under the coverslip and reach the edges. Allow the cyanoacrylate to dry completely. 23. Use cyanoacrylate to affix the head bar to the crown of the skull and allow it to dry completely. 24. Mix dental cement according to instructions. Use a plastic pick to spread the dental cement to cover the headbar and the remaining portions of the exposed skull. If a water immersion objective will be used in imaging experiments, use the cement to build a well around the edges of the coverslip, taking care not to cover the nares or the snout of the animal. 25. Once the animal has returned to a sternal and upright position, administer analgesics. Allow the animal to recover for at least 24 h.
While this preparation can be used for acute experiments immediately following window implantation, for best results, animals should recover for 1-3 days. During the surgical procedures, fluids can be ingested into the nasal cavities which can damage OSNs or occlude the ability to observe their functional responses. Allowing fluid to disperse and inflammation to subside improves calcium signal acquisition.

Results
Following a recovery period, both structural and functional fluorescence signals can be collected from the olfactory epithelium using 2-photon microscopy. The olfactory epithelium is highly vascularized, which will help in approximating the appropriate imaging depth, as well  as, repeatedly finding the same imaging location across experimental days ( Figure 2C). Importantly, care needs to be taken to distinguish between the OSN dendritic knob, found at the surface of the epithelium and the soma found deeper within the tissue. The transduction current in OSNs is partially carried by calcium and it is not known if the calcium dynamics at the dendritic knob are representative of spike-mediated calcium signals that propagate from the axon near the soma. In the dorsal epithelium, the orientation of OSNs is inverted, therefore the dendritic knobs will be found at a deeper imaging plane than the somata. Individual neurons can be visualized at rest; however, OSNs have variable spontaneous activity under baseline conditions, thereby limiting resting fluorescence signals from functional indicators if the spontaneous activity is low (Reisert, 2010;Connelly et al., 2013). Brief odor pulses can be delivered to drive OSN activity and aid in optimizing the imaging plane.
Once an optimal plane has been identified, the functional responses of dozens to hundreds of OSNs can be simultaneously measured. The density of neural activity in the olfactory epithelium varies as a function of odorant identity (Zak et al., 2020). If odorants that generate dense responses are used, care should be taken to account for background signals that may contaminate responses at individual cells. For all example experiments, the odorant Methyl tiglate was selected due to the high density of neural responses that it generates and for consistency across experiments.

Responses from individual OSNs in the olfactory epithelium
To capture functional responses from OSNs within the olfactory epithelium, 60-s pulses of the odorant Methyl tiglate were delivered to an anesthetized, but freely breathing mouse. The imaging session took place 3 days after the bone-thinning procedure to allow for surgically induced inflammation to subside. From a single imaging field, 410 OSNs ( Figure 3A) that were significantly odorant-modulated were identified (Figures 3B-D). OSNs were classified as significantly odorantmodulated if their peak response exceeded 2.5 standard deviations of the baseline noise. Although each of the OSNs had a clear stimulus-response, the waveform and response latency varied from cell to cell suggesting that multiple OSN subtypes are present in the imaging field (Figure 3E). With a diverse panel of odorants, populations of cells that express the same odorant receptor proteins could in principle be identified by their odorant tuning.
Many of these experiments were performed in anesthetized animals to exclude confounds associated with variable sniffing rates during a stimulus sampling period; however, it is also possible to perform similar experiments in awake animals. In these instances, functional OSN responses are locked to individual respiration cycles. Furthermore, when animals initiate sniffing bouts as indicated by a transient increase in respiration frequency, OSN responses are often directly modulated ( Figure 3F).
Odorant stimulations of 60 s give rise to the possibility of adaptation both at the level of receptor-ligand dynamics and intracellular calcium handling. To limit these potential effects, odorants were also delivered at a more physiologically relevant time scale of 2 s. Even at these brief stimulus durations, robust and repeatable OSN functional responses were observed ( Figure 3G). Together, these example data demonstrate that functional responses of OSNs can be reliably obtained using a thinned-bone procedure in combination with chronic window implantation.

Functional responses and window viability persists for weeks
To date, several studies have used a thinned-boned procedure to capture functional responses in OSNs (Iwata et al., 2017;Inagaki et al., 2020;Zak et al., 2020). A key advance here includes surgical implantation of a coverslip  above the thinned bone, which allows the same imaging field to be longitudinally followed across experimental days. To demonstrate the feasibility of repeatedly imaging the same subregion of the olfactory epithelium, the protocol above was used to create a stable chronic window. After allowing the window to stabilize for 3 days following implantation, the same subset of OSNs was imaged and their functional responses to the odorant Methyl tiglate were evaluated at time points spread across 3 weeks ( Figure 4A). The same populations of OSNs can be observed in the resting fluorescence images, while their functional responses are stable between imaging sessions ( Figure 4B). While the examples here demonstrate the ability to capture matched OSN responses across 21 days, the stability and clarity of the window can remain viable for several months, thereby allowing for extended imaging protocols.

Discussion
The approach described here has several advantages over acute preparations that allow for optical access to the olfactory epithelium (Iwata et al., 2017;Inagaki et al., 2020Inagaki et al., , 2021. First, the activity of OSNs can be measured longitudinally, allowing for matched comparisons across experimental conditions. Importantly, the application of cyanoacrylate under the glass coverslip prevents bone regrowth and thickening thereby allowing preparations to remain viable for experiments that can range from weeks to months. Furthermore, animals need not be anesthetized during imaging following a recovery period. This allows OSN activity to be measured within the context of natural respiration and sniffing dynamics in behaving animals. Combining behavioral readouts with measurements of the functional properties of individual neurons at the sensory periphery holds promise to provide new insight into sensory coding and computation at the earliest stages of the olfactory system. There are, however, a few limitations that should be noted. Due to the conserved zonal organization of the olfactory epithelium and the olfactory bulb (Ressler et al., 1993), there does not appear to be a substantial increase in the number of OSN subtypes available (Zak et al., 2020). Because the olfactory epithelium is organized on a dorsal-ventral axis with respect to OSN subtypes, many remain inaccessible (Figure 1B). From a technical perspective, the increased number of steps and the need to polish the thinned section of bone to maintain clarity creates more opportunities to damage the experimental preparation thereby potentially slowing workflows.
By implementing this approach, several challenging questions in sensory neurobiology can now be addressed. First transient disruptions of OSN activity profiles, like those imparted by the SARS-CoV-2 virus, can now be observed over the course of treatments or other manipulations to the supporting sustentacular cells which are affected by the virus. Furthermore, recent studies provide evidence of efferent modulation of the olfactory epithelium (Zhou et al., 2022), the experimental preparation described here may prove key to understanding neuromodulatory effects on peripheral sensory coding. Finally, the highly regenerative processes of the olfactory epithelium can be measured both in terms of sensory neuron turnover, but also changes in sensory coding properties with respect to the stimulus environment.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Harvard University.


PTH regulates osteogenesis and suppresses adipogenesis through Zfp 467 in a feed-forward, cyclic AMP-dependent manner

Conditional deletion of the PTH1R in mesenchymal progenitors reduces osteoblast differentiation, enhances marrow adipogenesis and increases zinc finger protein 467 (Zfp467) expression. In contrast, genetic loss of Zfp467 increased Pth1r expression and shifts mesenchymal progenitor cell fate towards osteogenesis and higher bone mass. In this study we hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. We report that PrrxCre Zfp467 but not AdipoCre Zfp467 mice exhibit an identical phenotype to the Zfp467-/-mice with high bone mass and greater osteogenic differentiation We also found that PTH suppressed Zfp467 expression primarily via the cyclic AMP/PKA pathway. Not surprisingly, PKA activation inhibited the expression of Zfp467 and gene silencing of Pth1r caused an increase in Zfp467 mRNA transcription. Dual fluorescence reporter assays and confocal immunofluorescence demonstrated that genetic deletion of Zfp467 resulted in higher nuclear translocation of p50 that binds to the P2 promoter of the Pth1r and increased its transcription. As expected, Zfp467-/- cells had enhanced production of cyclic AMP and increased glycolysis in response to exogenous PTH. Additionally, the osteogenic response to PTH was also enhanced in Zfp467 -/-calvarial osteoblasts, and the pro-osteogenic effect of Zfp467 deletion was blocked by gene silencing of Pth1r or a PKA inhibitor. In conclusion, our findings suggest that loss or PTH1R-mediated repression of Zfp467 results in a pathway that increases Pth1r transcription via p50 and thus cellular responsiveness to PTH, ultimately leading to enhanced bone formation.

9
almost evenly distributed in the cytoplasm and nucleus of +/+ cells but underwent partial 176 translocation to the nucleus in Zfp467 -/-cells (Fig. 4B). Zfp467 -/-cells also showed much higher 177 nuclear protein level of both p50 and GATA1 (Fig. 4C, D). 178 179 p50-Relb heterodimer may drive greater Pth1r transcription in Zfp467-/-cells 180 In order to confirm whether p50 or GATA1 could activate the specific P1 or P2-2 Pth1r promoter, 181 we co-transfected MC3T3-E1 cells with P1 and P2-2 dual-luciferase reporter and GATA1, p50 182 overexpression plasmids. Only the p50 overexpression group was able to significantly activate the 183 P2-2 promoter (Fig. 5A). ChIP results showed that the DNA was properly sheared and IP was 184 successfully conducted (Fig. 5B). ChIP-qPCR results showed that the first two parts of P2 were 185 properly enriched in our IP product (>0.5%) (Fig.5-figure supplement A), and the first part of P2 186 was approximately 20 fold more highly enriched in our p50 IP product than IgG (Fig. 5C, Fig.5-187 figure supplement B); this indicated that p50 binds to the P2 promoter, especially at the first 200 188 bp site. Subsequently, we treated COBs and BMSCs with p50 siRNA and found that p50 knock-189 down could significantly inhibit the expression of Pth1r in both +/+ and -/- COBs and BMSCs. 190 Importantly, p50 knock-down in -/-cells reverts the levels of Pth1r to the levels seen in +/+ cells 191 ( Fig. 5D-F). 192 In order to determine whether p50 could bind to the Pth1r P2-2 promoter directly, we performed 193 DNA pulldown assay using biotin-labeled Pth1r P2 promoter as a probe. As shown in Fig. 6A, the 194 biotin-Pth1rP2 group showed a specific band in both MC3T3-E1 nuclear extracts and purified p50 195 protein, suggesting a direct physical interaction between p50 and Pth1r P2 promoter. However, we 196 noticed that p50 does not have a transcriptional activation domain, so p50 must heterodimerize 197 10 with other transcription factors in order to increase gene transcription. Using String database and 198 checking published studies, we found 8 candidate that might heterodimerize with p50 to regulate 199 gene transcription: NFYC, NPAS1, Rel, AKAP8, RelA, RelB, ANKRD42 and HDAC1. Using 200 siRNA, we knocked down all these potential p50 partners (Fig. 6B), but the upregulated Pth1r 201 induced by p50 overexpression could only be dampened by Npas1 and Relb siRNA (Fig. 6C). 202 Further co-immunoprecipitation results confirmed that p50 could heterodimerize with RelB only 203 (Fig. 6D), which suggested p50-Relb heterodimer may drive greater Pth1r transcription in 204 Zfp467-/-cells. 205 206

Zfp467 -/-cells have increased PTH signaling and higher extracellular acidification rates 207 (ECAR) 208
As the PKA pathway is considered to be one of the major downstream pathways of the PTH 209 signaling network, it would be important to know whether -/-cells have higher PKA activation 210 levels due to the higher expression of PTH1R. PTH increased cAMP within 10 minutes ,measured 211 by ELISA in +/+ COBs, and -/-cells had a higher levels of cAMP expression than +/+ cells 212 (p=0.0007 for vehicle, p<0.0001 for 50nM, p=0.0008 for 100nM). In addition, -/-BMSCs also had 213 significantly higher levels of intracellular cAMP after 10-60 min exposure of PTH (p<0.0001 for 214 10 min-100nM, p=0.0341 for 30 min-100nM, p<0.0001 for 60 min-100nM) (Fig. 7A, B). 215 Importantly, -/-COBs and BMSCs showed a greater magnitude of increase of cAMP after PTH 216 treatment (p=0.0053 for interaction in COBs, p=0.0010 for interaction in BMSCs), which resulted 217 from higher PTH1R in -/-cells. Additionally, as CREB was one of the major downstream targets 218 of PKA, we found higher phosphate ratio of CREB in -/-COBs and BMSCs whereas the total 219 11 protein level of CREB showed no difference (Fig. 7C, D). 220

221
In a previous study, PTH was shown to enhance aerobic glycolysis, which is a major source of 222 ATP for osteoblast differentiation(16). We measured the oxygen consumption and extracellular 223 acidification in both COBs and BMSCs pre-osteogenic differentiation and 3 days after osteogenic 224 differentiation. Cellular respiration measurements showed that  BMSCs had significantly increased ECAR, but no difference was found for pre-differentiated 226 COBs in respect to either ECAR or oxygen consumption rate (OCR) (Fig. 7E, F). However, both -227 /-COBs and BMSCs cells had significantly higher ECAR levels after 3 days of osteogenic 228 differentiation, although no difference was found regarding OCR between genotypes (Fig. 7G, H). 229 These data suggest that -/-COBs or BMSCs may have higher glycolysis as a result of higher 230 PTH1R expression. 231 232 Zfp467-/-cells showed increased sensitivity to PTH and enhanced pro-osteogenic as well as 233

anti-adipogenic effects 234
To determine the sensitivity of Zfp467-/-cells to PTH, we treated COBs with osteogenic 235 differentiation media and PTH for 7 or 14 days simultaneously. A dose response to PTH led to a 236 significant increase in ALP staining in -/-COBs, and in +/+ cells. Furthermore, 100nM PTH in -/-237 cells produced remarkably higher positive-stained cells than vehicle group (p=0.0001) while there 238 was no statistical significance seen among +/+ groups (p=0.6536) (Fig. 8A, B). Importantly, -/-239 COBs showed a more significant response to PTH regarding ALP staining (p=0.0201) (Fig. 8B). 240 Alizarin red staining showed a parallel trend as ALP staining; an increase in PTH dose resulted in 12 an increase in mineralization for -/-COBs only (Fig. 8A). 242 In osteogenic media, calvarial progenitors can differentiate into adipocytes. We noted significantly 243 less lipid droplets in -/-adipocytes cultured in osteogenic media compared to untreated +/+ 244 samples (Fig. 8C). Additionally, a reduction in adipogenesis was observed with PTH exposure at 245 50nM and 100nM in both +/+ and -/-cells, and -/-cells showed much less lipid droplet formation 246 compared to +/+ group (Fig. 8C, D). However, the magnitude of decrease after PTH treatment is 247 almost identical in +/+ and -/-cells (Fig. 8D). These results were then confirmed by qRT-PCR 248 after 7 days osteogenic differentiation which indicated higher expression of osteogenic 249 differentiation related genes such as Alp, Sp7, Rankl and Igf-1 in -/-compared to +/+ COBs with 250 PTH exposure (Fig. 8E, F). It is noteworthy that there is a statistically significant interaction 251 between genotype and PTH treatment, suggesting an increased sensitivity to PTH in -/-cells. To determine whether the increase in osteogenic differentiation seen in Zfp467-/-cells is due to 256 higher PTH1r levels, we knocked down Pth1r by siRNA and found that Pth1r knock down led to 257 decreased osteogenic differentiation in both +/+ and -/-COBs. Although -/-COBs still show 258 slightly higher Alp staining (Fig. 9A), Pth1r knock down in -/-cells dampens the increase in Alp 259 and Sp7 gene expression during osteogenic differentiation compared to +/+ cells, indicating that 260 this increase is associated with the up-regulation of Pth1r seen in -/-cells (Fig. 9B). 261 Furthermore, a PKA inhibitor could also significantly decrease osteogenic differentiation in COBs 262 (Fig. 9C). The staining results showed ALP activity was much higher in -/-cells in the absence of 13 the PKA inhibitor, but no difference was found with the PKA inhibitor between genotypes (Fig.  264   9C). In addition, we observed that with the PKA inhibitor, upregulated osteogenic genes in -/-265 cells including Alp and Sp7 could be totally reversed (Fig. 9C). Similarly, treating COBs with the 266 PKA inhibitor during PTH treatment simultaneously for 7 days led to a suppression of osteogenic 267 differentiation in both +/+ and -/-cells ( Fig. 9-figure supplement). qPCR results showed that 268 PKA inhibitor could totally reverse the upregulation of Alp, Sp7 and Rankl in PTH treated -/-cells 269 ( Fig. 9-figure supplement A, B). We then next asked what were the downstream targets of Zfp467. In an unbiased analysis, we used 273 RNAseq in pre-and post-differentiated calvarial osteoblasts from +/+ and Zfp 467 -/-cells to 274 assess potential regulatory pathways and differentially expressed genes ( Fig. 10A-D). The PI 3-K 275 and MAPK signaling pathways were differentially up regulated in Zfp467-/-cells whether pre-or 276 post-differentiated when compared to WT (Fig. 10D). There were several highly expressed genes 277 in the -/-cells related to osteogenesis, including Wdfy1, Sox10, and Ngfr ( Fig. 10B) These results 278 were confirmed by qRT-PCR (Fig. 10E). When Zfp 467 was over-expressed in MC3T3-E1 cells 279 those three genes were significantly suppressed relative to Gfp overexpression. In this paper we characterize the role of Zfp467 in the molecular, cellular and biochemical 283 responses to PTH in osteoblast progenitors. Based on our data, we propose a novel pathway 284 by which PTH can enhance its own anabolic actions in osteoblast progenitors by repressing 285 Zfp467, a negative regulator of PTH1R expression. Zfp467 was originally isolated from 286 mouse hematogenic endothelial LO cells as an OSM-inducible mRNA, which encodes protein 287 ZFP467(17), also called EZI. Zinc finger motifs are involved in protein-protein interactions 288 and protein-DNA binding (10,18,19). Based on the zinc finger domain, zinc finger proteins are 289 classified into C2H2, C3H and C4(20). ZFP467 belongs to C2H2 zinc finger protein family 290 whose zinc finger domain consists of two cysteine and two histidine residues. ZFP467 was 291 initially found to cooperate with STAT3 and augment STAT3 activity by enhancing its nuclear 292 translocation in a kidney cell line(17). ZFP467 is expressed ubiquitously and may be an 293 important mediator of bone marrow cell differentiation into the adipogenic or osteogenic 294 lineages as well as functioning in other tissues in distinct ways (12,13,21). 295 In our previous work we found that deletion of pth1r in mesenchymal cells resulted in low 296 bone mass, high marrow adiposity, and upregulation of Zfp467(8). In contrast, the absence of 297 Zfp467 resulted in a significant increase in trabecular bone volume fraction, accompanied by 298 a marked reduction in peripheral and marrow adipose tissue and improved glucose 299 tolerance(14). Most importantly for the current study, whole bone marrow gene expression by 300 qRT-PCR revealed a ~40% increase in Pth1r expression and greater protein levels in the 301 Zfp467 -/-mice compared to controls(14). Therefore, we hypothesized that PTH1R and 302 ZFP467 could be involved in a feedback loop whereby the suppression of Zfp467 mediated by 303 PTH leads to an increase of PTH1R, and therefore an enhanced response to PTH treatment. 304 However, due to the global nature of the Zfp 467 deletion, we could not exclude a cell non-305 autonomous effect. In the present study therefore we tested that hypothesis both in vivo and 306 in vitro, and sought to determine the cellular and biochemical mechanisms involved in this 307 15 novel regulatory pathway. 308 First, we were able to show that conditional deletion of Zfp 467 by PrrxCre led to an increase 309 in osteogenesis and bone formation, a finding that mirrors our results with the global deletion 310 of Zfp 467. On the other hand, using an AdipoCre driven system, we found no effect of 311 conditional deletion in adipocytes on body composition, fat mass, bone mass or total weight. 312 Taken together these data would suggest that Zfp467 is an early mesenchymal transcriptional 313 factor that regulates lineage allocation in early osteoblast but not adipocyte progenitors. Second, in 314 this report, we found constitutive up-regulation of Zfp467 when we genetically knocked down 315 Pth1r in calvarial osteoblasts and bone marrow stromal cells. In addition, much like the study of 316 Quach et al. (12), we noted that acute PTH treatment could significantly suppress gene expression 317 of Zfp467 in both COBs and BMSCs, and the suppression could be partly rescued by both a PKA 318 pathway inhibitor and a PKC inhibitor (12). Similarly, Forskolin, a PKA pathway activator could 319 also inhibit the expression of Zfp467, with a more sustained effect. These data indicated that PTH 320 might suppress Zfp467 expression via activation of the PTH1R through predominantly PKA 321

pathways. 322
To test the validity of our hypothesis, we investigated how deletion of Zfp467 affected the 323 expression of the PTH1R. We first examined which transcript of Pth1r was upregulated and 324 further confirmed that via dual-fluorescence reporter assay that both P1 and P2 promoters of Pth1r 325 were activated in Zfp467 null cells; however P2 was more activated than P1. Using three different 326 transcription factor prediction databases including PROMO, JASPAR and Animal TFDB, we 327 found several candidate transcription factors that might be involved in the regulation of Pth1r in 328 Zfp467 -/-cells via activation of the P2 promoter. After overexpressing or knocking down each 329 16 candidate transcription factor, we found that only p50 could up-regulate Pth1r via activation of the 330 P2-2 Pth1r promoter. Further confocal immuno-florescence and nuclear protein detection 331 indicated that the nuclear translocation of p50 was much higher in Zfp467-/-cells. Moreover, 332 ChIP-qPCR results showed that p50 could bind to the P2 promoter of the Pth1r. Taken together, 333 these data suggested that the deletion of Zfp467 resulted in higher nuclear translocation of p50 334 which bound to the P2 promoter of Pth1r and promoted its transcription. 335 p50 is one of the DNA binding subunits of the NF-kappa-B (NFκB) protein complex. NFκB is a 336 transcriptional regulator that is activated by various stimuli including cytokines, bacteria and 337 oxidation. The NFκB pathway is involved in several biological processes including inflammation, 338 bone resorption, aging and cancer (22). Activated NFκB translocates into the nucleus and 339 stimulates the expression of an array of genes. However, we found no previous studies that p50 340 may regulate the gene expression of Pth1r or other osteogenic related genes. Nevertheless, it is 341 clear that p50 is involved in osteoclastogenesis and bone resorption, hence it plays a significant 342 role in bone remodeling. It is also conceivable that p50 could associate with histone deacetylase-1 343 (HDAC1) or be regulated by a PKA catalytic subunit which is also downstream of PTH signaling 344 (23). Moreover, it is likely that other proteins like RelB that might bind to p50 and enhance its 345 effect on the transcriptional regulation of Pth1r play a critical role in PTH mediated osteogenesis. 346 Osteoblasts require substrates for energy utilization during collagen synthesis and mineralization. 347 Previous reports by Lee et al. and Guntur et al. demonstrated that glycolysis is a major source of 348 ATP for differentiating osteoblasts(9,16). Consistently, we found that Zfp467 -/-cells showed 349 higher cAMP levels in response to PTH as well as higher glycolytic activity (i.e. ECAR). COBs 350 from Zfp 467-/-mice also showed greater differentiation in osteogenic media but less 351 17 differentiation into adipocytes compared to controls. Higher rates of osteogenesis, enhanced Rankl 352 and Igf1 expression, increased glycolysis and decreased adipogenesis are likely related to greater 353 activation of the PTH1R, possibly due to its higher endogenous expression level. 354 Since it was reported that the downstream effects of PTH could be partially blocked by PKA 355 inhibitors (24), we used Pth1r siRNA and PKA inhibitors to block the effect of PTH1R. We 356 confirmed that PTH1R and its downstream pathways were involved in the deletion of Zfp467  Despite uncovering a novel regulatory circuit through p50, we recognize there are some 371 limitations to our study. First, it should be noted that we also found that Zfp467-/-cells have 372 higher nuclear translocation of GATA1 and overexpression of GATA1 could promote Pth1r 373 18 expression. However overexpression of GATA1 failed to activate the P2-2 promoter of Pth1r. 374 Hence, we believe that GATA1 might be another of the regulating transcription factors between 375 ZFP467 and Pth1r, but it likely regulates Pth1r expression via binding sites other than those 376 present on P2-2. 377 Second, we have not determined how Zfp467 binds with p50-RelB heterodimer, and we have not 378 identified the exact genomic sequence required for p50-RelB heterodimer binding at the P2 379 promoter binding site. Further studies will be needed to address this important mechanistic 380 question. Hence the precise mechanism of action of Zfp467, and the downstream consequences of 381 p50-RelB heterodimer binding on the presumed switch between adipocytes and osteoblasts is still 382 not fully defined. 383 Last, to our knowledge there must be a break on the activation of a feed forward system, but we 384 have to acknowledge that we have not delineated whereby the fast forward system interacts with 385 β-Arrestin or any other turns off signaling. 386 In summary, taken together, we demonstrated the importance of the zinc finger protein ZFP467 for 387 lineage allocation in vitro and in vivo, as well as responsiveness to PTH in osteoblast progenitors. 388 Our data also support a novel feed-forward regulatory loop whereby suppression of Zfp467 389 mediated by PTH and its downstream PKA pathway leads to an increase of PTH1R via p50, and 390 subsequent enhanced responsiveness to PTH treatment. These findings have significant 391 implications for our understanding of the anabolic effects of PTH on bone. Zfp467 fl/fl on the C57BL/6 J background was generated by Cyagen ( Fig. 1 supplement 1). Exons 407 2~4 were selected as conditional knockout region (Transcript: Zfp467-001 408 ENSMUST00000114561). The targeting vector, homology arms and cKO region were generated 409 by PCR using Bacterial Artificial Chromosome (BAC) clone RP24-144J8 and RP23-24K23 from 410 the C57BL/6J library as template. To generate mice lacking Zfp467 in limb mesenchymal stem 411 cells, Prrx1Cre; Zfp467 fl/fl were generated by crossing Prrx1Cre transgenic mice to Zfp467 fl/fl mice 412 (Prx-Cre Zfp467 fl/fl ) and · · · · · · · · · · · · · · · · · · · · · · · · · fl/fl mice were used as controls. To generate mice lacking Zfp467 in

Committee. 424
No statistical methods were used to predetermine sample size. Mice label and measurements were 425 performed by two independent researchers and selected at random (by cage) into following 426 experiments. Masking was used during data collection and data analysis. Animals with ulcerative 427 dermatitis or other diseases were excluded from the study. 428

Dual-energy X-ray Absorptiometry (DXA) 429
Whole body composition evaluation of the head was performed using the PIXImus densitometer 430 (GE-Lunar, Fairfield, CT, USA). The PIXImus was calibrated daily with a phantom provided by 431 the manufacturer. 432

Brüttisellen, 435
Switzerland) was used to assess the trabecular and cortical bone microarchitecture, volume and 436 mineral density in mouse femurs. Scans were acquired using a 10.5 μm 3 isotropic voxel size, 70 437 kVp peak x-ray tube intensity, 114 mA xray tube current, 250 ms integration time, and were 438 21 subjected to Gaussian filtration and segmentation. All scans were analyzed using manufacturer

Marrow adipose tissue quantification by osmium tetroxide staining and μCT 452
At the time of sacrifice, tibiae were isolated and placed into 10% neutral buffered formalin 453 overnight at 4°C. Soft tissue was carefully removed to ensure that the fibula remained intact and 454 the bones were washed under continuous cold PBS for one hour, then stored in PBS at 4°C. 455 Quantification and visualization of marrow adipose tissue was performed as described 456 previously(32). Briefly, bones were decalcified in 14% EDTA (pH 7.4) for 14 days, with EDTA 457 changes every 3-4 days. Bones were then washed for 10 minutes in PBS (3 times) and stained with 458 a 1:1 mixture of 2% aqueous osmium tetroxide (cat# 23310-10, Polysciences, Inc., Warrington, 459 PA, USA) and 5% potassium dichromate for 48 hours. Stained bones were then washed with PBS 460 22 (pH 7.4) for 5 hours (3 times), and subsequently scanned by μCT. Bone marrow adipose tissue 461 (BMAT) content was calculated by determining the whole volume of second osseous center of 462 tibiae. 463

Primary cells isolation 464
The generation of Zfp467-/-and wild-type mice was previously described. Calvaria osteoblasts 465 (COBs) were isolated from calvarias of 3-5-day old +/+ and -/-neonates as described in the Cytoplasm and nuclear protein were extracted using Nuclear and Cytoplasmic Extraction Reagent 489 Kit (Thermo Scientific, MA, US). Antibodies used for western blot and Chromatin 490 Immunoprecipitation (ChIP) or co-Immunoprecipitation (co-IP) were listed in Table 1  DNA fragments were used as templates for PCR amplification. ChIP-PCR primers sequences were 516 listed in Table 2. PCR products were used for nucleic acid electrophoresis to avoid unspecific 517 25 amplification. 518 DNA pulldown assay was performed according to previously reported protocol (36). 5' biotin-519 modified Pth1r P2-2 dsDNA probe was generated using PCR with oligonucleotide primers 520 modified at its 5' end by Sangon Biotech (Shanghai, China). Nuclear protein were extracted using 521 Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, MA, US). p50 purified 522 protein was purchased from Proteintech (Rosemont, US). Briefly, the Pth1r P2-2 or oligo probe 523 was incubated with streptavidin-coupled Dynabeads (Invitrogen, US) at room temperature for 1 524 hour to generate probe-bound Dynabeads, and then the probe-bound Dynabeads were incubated 525 with MC3T3-E1 nuclear extracts or p50 purified protein at 4 °C overnight. The protein bound to 526 the probe and beads were eluted and used for gel electrophoresis.  Carlsbad, CA) was used for normalization. 546

Osteogenic differentiation and related measurements 547
In vitro osteoblast differentiation and measurement were done according to previously published 548 protocols(14). Briefly, BMSCs and COBs were plated at a density of 1x10 6 /well in 6-well plates. 549 When cells reached around 80% confluency, osteogenesis induction began using osteogenic 550 induction media which consisted of complete αMEM (MEM, 5% fetal bovine serum, and 1% 551 penicillin/streptomycin), 50ug/mL ascorbic acid, and 8mM beta-glycerophosphate (both were 552 27 purchased from Sigma, St. Louis, MO)]. MEM and penicillin/streptomycin were purchased from 553 Life Technologies (Carlsbad, CA), FBS was purchased from VWR (Radmor, PA). Medium was 554 changed every other day until cells were ready to stain for alkaline phosphatase (ALP) and alizarin 555 red (ARS) to assess osteoblasts and mineralization, respectively, around d7 and d14 after 556 differentiation. Additionally, RNA was extracted on day 7 after differentiation for real time PCR 557 analysis. 558

Alkaline Phosphatase and Alizarin Red Staining 559
Alkaline phosphatase staining was performed using ALP kit obtained from Sigma (St. Louis, MO) 560 according to the manufacturer's instructions at d4 and d7 after osteogenic differentiation for 561 BMSCs and COBs, respectively. ARS (Sigma, St. Louis) staining was done using 1% solution at 562 pH 4.2 at d14 after osteogenic differentiation for both BMSCs and COBs. Briefly, after fixation, 563 cells were stained for 30 minutes with ARS solution at room temperature. Cells were then washed 564 a couple of times with water before they were visualized under the microscope (Leica DM IRB, 565 TV camera). Five randomly fields were chosen to capture images using the ZEISS Efficient 566 Navigation, blue edition camera (Bloomfield, CT) per treatment group for quantification. 567

Oil Red O (ORO) staining 568
Differentiated adipocytes were stained using ORO solution at d9 after adipogenic diff. Briefly, 569 cells were fixed with 10% neutral buffer formalin (Sigma, St. Louis). After that, they were washed 570 with 60% isopropanol (Sigma, St. Louis, MO) before stained with ORO solution (Sigma, St. 571 Louis, MO) for 15 minutes at room temperature. Cells were then washed a couple of times with 572 water before they were visualized and pictures were taking using the Zeiss microscope. For 573 quantification, lipid from adipocyte droplets was extracted using isopropanol and the absorbance 574 28 was measured at 490 nm using a plate reader (MRX Dynex Technologies, Chantilly, VA). 575

RNA-seq and Analysis 576
RNA-seq was performed on samples from COBs isolated from Zfp467+/+ and -/-mice, pre-or 577 after-4 days' osteogenic differentiation. RNA-seq analysis was performed by BGI Group (UK, 578 China). Samples were de-identified, and the analysis was blinded to assignment. We employed 579 DAVID 6.8 to perform the functional annotation (Cellular Component [CC]) and GSEA 580 enrichment plots for related pathways. Protein coding genes were assessed with p<0.05, fold 581 change > 2.0 or < -2.0. The enriched CC was evaluated using a false discovery rate (FDR, 582 Benjamini-Hochberg method) of 0.1 and DEG number. We have uploaded our original 583 sequencing data to Sequence Read Archive database (PRJNA877934, 584 http://www.ncbi.nlm.nih.gov/bioproject/877934). 585

Statistical Analysis 586
All data are expressed as the mean ± standard deviation (SD) unless otherwise noted. Results were 587 analyzed for statistically differences using Student's t-test between two groups or 2-way ANOVA 588 followed by Bonferroni's multiple comparison post hoc test among three or more groups where 589 appropriate. All statistics were performed with Prism GraphPad 7.0 statistical software (GraphPad 590 Software, Inc., La Jolla, CA). Values of p<0.05 were considered statistically different. 591 conditional knockout region. The targeting vector was generated by PCR using BAC clone RP24-728 144J8 and RP23-24K23 from the C57BL/6J library as template. 729 shown as mean ± SD by unpaired Student's t test, n=7-10 per group. 733 BMSCs. Data shown as mean ± SD by one-way ANOVA, n=3 independent experiments for each 738 group. (C) qPCR results· of Zfp467+/+ in COBs with 2 hours PKA or PKC inhibitor treatment 739 prior to 10min of 100 nM PTH exposure, PKA but not PKC inhibitor was able to rescue the 740 suppression of Zfp467 induced by PTH. Data shown as mean ± SD by one-way ANOVA, n=3 741